Beta-catenin abnormalities and associated insulin-like growth factor overexpression are important in phyllodes tumours and fibroadenomas of the breast

The aim of this study was to assess the expression of IGF-I and IGF-II in phyllodes tumours and fibroadenomas and to see if there is any correlation between nuclear beta-catenin expression and IGF-I and IGF-II expression in these tumours. In a previous study, it has been shown that Wnt signalling is important in the pathogenesis of phyllodes tumours of the breast. Epithelial Wnt5a overexpression and stromal Wnt2 overexpression were associated with abnormal, nuclear localization of beta-catenin in the stromal cells of these tumours. However, not all tumours with beta-catenin accumulation showed Wnt overexpression. One other possible cause of beta-catenin accumulation is overexpression of insulin-like growth factors (IGFs), as both IGF-I and IGF-II have been shown to stabilize beta-catenin. In this study, 30 fibroadenomas of the breast were assessed for beta-catenin expression using immunohistochemistry and the results were compared with previous data from 119 phyllodes tumours. In situ hybridization was used to assess IGF-I and IGF-II expression in 23 phyllodes tumours and 16 fibroadenomas. Nineteen phyllodes tumours (83%) showed widespread overexpression of IGF-II and 5/23 (22%) showed widespread overexpression of IGF-I. IGF-I expression correlated with nuclear beta-catenin staining in phyllodes tumours. Malignant phyllodes tumours showed no or little IGF-I expression. There was a degree of nuclear beta-catenin expression in the stroma (weak in 33%, moderate in 27%, and strong in 40%) in all fibroadenomas and nuclear beta-catenin staining correlated with IGF-I overexpression. Extensive IGF-II overexpression was also found in the majority of fibroadenomas (12/16). These results support the hypothesis that IGF-I and IGF-II overexpression may be important in the pathogenesis of fibroepithelial neoplasms of the breast and that IGF-I overexpression is likely to be contributing to the nuclear beta-catenin localization observed in the tumours.     

Endothelin-1 expression correlates with atypical histological features in mammary phyllodes tumours

BACKGROUND AND AIMS: Endothelin-1 expression is increased in infiltrating duct carcinoma and is associated with larger tumour size, higher histological grade and lymphovascular permeation. This has not been evaluated in phyllodes tumours, which are uncommon fibroepithelial lesions with potential for local recurrences or distant metastasis. While the grading of phyllodes tumours depends on a combination of histological parameters, prediction of their behaviour remains difficult. METHOD: A large series of 461 phyllodes tumours (291 benign, 115 borderline malignant and 55 frankly malignant) were evaluated for endothelin-1 expression in both the epithelial cells and stromal cells by immunohistochemistry; results were correlated with the tumour grade. RESULTS: For benign phyllodes tumours, the epithelial staining of endothelin was negative, weak, moderate and strong in 6%, 26%, 15% and 53% of cases respectively; results were 4%, 18%, 19% and 59% respectively for borderline and 6%, 18%, 6% and 70% respectively for frankly malignant tumours. For the stromal staining, the negative, weak, moderate and strong staining was 32%, 19%, 18% and 31% respectively for benign phyllodes, 24%, 13%, 10% and 53% respectively for borderline and 8%, 16%, 17% and 59% respectively for frankly malignant tumours. There was correlation between epithelial and stromal staining, and the stromal staining correlated with histological features of stromal cellularity, stromal cell nuclear pleomorphism, margin status and stromal overgrowth. CONCLUSION: These observations suggest a close relationship between the epithelial and stromal elements in phyllodes tumours; endothelin may play a significant role in the malignant progression of phyllodes tumours.     

Malignant phyllodes tumours show stromal overexpression of c-myc and c-kit

Phyllodes tumours are fibroepithelial neoplasms of the breast, the stroma of which can undergo malignant progression to sarcoma. The frequency of malignant lesions varies in different series from 5% to 30%. The aim of this study was to elucidate potential molecular mechanisms in the progression to malignancy in phyllodes tumours. c-myc and c-kit were studied at the protein, RNA(c-myc only) and DNA level. We chose to study c-myc as we have previously shown that Wnt signalling is important in benign, but not malignant, phyllodes tumours. If c-myc is constitutively activated in malignant tumours, this may provide an explanation for why the Wnt pathway is no longer important in these tumours. c-kit is a membrane-bound tyrosine kinase receptor and overexpression is characteristic of gastrointestinal stromal tumours. A previous report suggested that this may also be the case in malignant phyllodes tumours, and we wished to confirm this. We assessed expression of c-myc and c-kit in 30 phyllodes tumours (10 malignant) using in situ hybridization (c-myc) and immunohistochemistry (c-myc and c-kit). 9/10 malignant tumours showed c-myc expression in the stroma, compared to 7/20 benign tumours (p = 0.006, Fisher's exact test). Stromal c-kit expression was found in 5/10 malignant tumours, compared to 1/20 benign tumours (p = 0.008, Fisher's exact test). One tumour had high-level amplification of c-myc, but we found no evidence of mutations of c-kit. We hypothesize that the overexpression of c-myc may drive stromal proliferation in malignant phyllodes tumours, and that c-kit overexpression contributes to the growth of these lesions. c-kit may also be a new therapeutic target in these tumours.     

p16 and pRb immunohistochemical expression increases with increasing tumour grade in mammary phyllodes tumours

Karim R Z, Gerega S K, Yang Y H, Spillane A, Carmalt H, Scolyer R A & Lee C S
(2010) Histopathology 56, 868–875
p16 and pRb immunohistochemical expression increases with increasing tumour grade in mammary phyllodes tumours Aims: Control of cell cycling and proliferation is critical to the development of neoplasia and may play a role in the pathogenesis of phyllodes tumours (PTs). This study aimed to evaluate the immunohistochemical expression of certain proteins from the G₁/S transition of the cell cycle in a cohort of PTs, to determine their role in tumour pathogenesis and to identify any associations with patient outcome. Methods and results: Sixty‐five PTs (34 benign, 23 borderline and eight malignant) diagnosed at a single institution between 1990 and 2006 were analysed. Immunohistochemistry for p16, pRb, cyclin D1 and Ki67 was performed. Expression of the following markers increased significantly with tumour grade: stromal nuclear and cytoplasmic p16 (P = 0.01 and 0.002, respectively), stromal and epithelial pRb (P = 0.000 000 06 and 0.004, respectively), and stromal and epithelial Ki67 (P = 0.03 and 0.04, respectively). Epithelial pRb scores of 7 (range 0–7) were significantly associated with reduced disease‐free survival (DFS) compared with scores of <7 (P = 0.0009). No relationship was found between cyclin D1 expression in either the epithelium or the stroma, and grade or DFS. Conclusions: The results suggest that alterations at the G₁/S transition of the cell cycle play an important role in the progression of PTs.     

Malignant phyllodes tumours of the breast display increased stromal p53 protein expression

AIMS: To determine the variation in p53 protein expression in phyllodes tumours and fibroadenomas of the breast. METHODS AND RESULTS: Fifteen phyllodes tumours (six malignant, nine benign) and 20 fibroadenomas were examined for p53 expression by immunohistochemistry. Five of the six malignant phyllodes tumours showed moderate or strong p53 positivity at sites of peri-epithelial stromal condensation and atypia. All 20 fibroadenomas, nine benign phyllodes tumours and one malignant phyllodes tumour showed either negativity or focal weak nuclear positivity of scattered stromal cells. CONCLUSIONS: Increased p53 immunoreactivity is present in malignant phyllodes tumours in contrast to benign phyllodes tumours and fibroadenomas. Malignant phyllodes tumours display a distinctive pattern of p53 immunostaining which may be of diagnostic value. These findings suggest that p53 protein may be important in the progression of benign to malignant phyllodes tumours.     

Analysis of the progression of fibroepithelial tumours of the breast by PCR-based clonality assay

Fibroadenoma and phyllodes tumour of the breast are both fibroepithelial tumours. Although progression to epithelial malignancy has been described, the behaviour of most fibroadenomas is benign. Phyllodes tumours, on the other hand, can display locally destructive growth and can even metastasize. A relationship between the two tumours has been suggested in the literature. This study investigated the clonality of both the stroma and the epithelium of these fibroepithelial tumours and attempted to construct a model in which fibroadenoma can progress in both an epithelial and a stromal direction. Fibroadenomas (n=25) and phyllodes tumours (n=12) were selected for analysis. Tissue was microdissected and analysed for clonality using a polymerase chain reaction (PCR)-based assay targeted at an X-linked polymorphic marker, the human androgen receptor gene (HUMARA). Nineteen fibroadenomas and nine phyllodes tumours could be analysed. Normal-appearing epithelium, hyperplastic epithelium, and stroma removed from fibroadenomas were polyclonal. As expected, carcinoma in situ (CIS) removed from four fibroadenomas was monoclonal. Three areas of apparent stromal expansion within fibroadenoma were monoclonal, suggesting stromal progression. Mostly, the stroma of phyllodes tumours was monoclonal and the epithelium polyclonal. In two cases, however, the epithelium seemed to be monoclonal, whereas in three other cases the stromal component was polyclonal. These findings indicate that fibroadenoma can progress in an epithelial direction to CIS and in a stromal direction to phyllodes tumour.     

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.     

Predictors of phyllodes tumours on core biopsy specimens of fibroepithelial neoplasms

Jara‐Lazaro A R, Akhilesh M, Thike A A, Lui P C‐W, Tse G M‐K & Tan P H
(2010) Histopathology 57 , 220–232 Predictors of phyllodes tumours on core biopsy specimens of fibroepithelial neoplasms Aims: To establish histological and biological parameters that can predict phyllodes tumours on core biopsy specimens of indeterminate fibroepithelial neoplasms. Methods and results: Core biopsy specimens of fibroepithelial lesions diagnosed at the Department of Pathology, Singapore General Hospital from 2002 to 2007 were reviewed. Cases in which phyllodes tumour was favoured, or could not be ruled out, were evaluated for stromal cellularity/distribution, nuclear atypia and mitoses, stromal overgrowth, epithelial fronding, epithelial hyperplasia, configuration of lesional edge, presence of pseudoangiomatous stromal hyperplasia and of adipose tissue. Antibodies to Ki67, topoisomerase IIα, CD34, CD117 and Bcl‐2 were applied to sections subjected to immunohistochemistry using the streptavidin–biotin method. Findings were correlated with subsequent excisions. Of 261 core biopsy specimens of fibroepithelial lesions, 98 (37%) comprised cases in which phyllodes tumour could not be excluded and 57 (58%) had subsequent open surgical excisions. Marked stromal hypercellularity (5/5; 100%) and nuclear atypia (1/1; 100%), stromal overgrowth (17/17; 100%), mitoses ≥2/10 high‐power fields (18/18; 100%) and ill‐defined lesional borders (16/16 phyllodes tumours; 100%) were features in core biopsy specimens that exclusively predicted phyllodes tumour on excision. Moderate stromal hypercellularity (20/27 phyllodes tumours; 74%), stromal overgrowth, moderate nuclear atypia (14/16 phyllodes tumours; 87%), pseudoangiomatous stromal hyperplasia (19/23 phyllodes tumours; 83%) significantly correlated with their subsequent excisions. Immunohistochemical markers Ki67 ≥5% and topoisomerase IIα≥5%, and reduced or patchy CD34 on core biopsy specimens correlated significantly with a diagnosis of phyllodes. Conclusions: Stromal hypercellularity, combined with key histological features and immunohistochemical markers Ki67, topoisomerase IIα and CD34, reinforced by clinical findings, can predict phyllodes tumours on core biopsy specimens.     

Expression of non-membranous beta-catenin and gamma-catenin, c-Myc and cyclin D1 in relation to patient outcome in human colon adenocarcinomas

Non-membranous beta-catenin and gamma-catenin, c-Myc and cyclin D1 are key participants in the Wnt cell signalling pathway, in which aberrancies have been associated with malignant cell transformation. We assessed the independent prognostic value of these proteins in a clinical material. Tumours from a series of 162 patients operated on for Dukes' stage A, B and C colonic adenocarcinomas were analysed using semiquantitative immunohistochemistry and the results were related to patient outcome. Patients expressing nuclear beta-catenin in the primary tumour showed reduced survival compared to other patients (log rank p=0.028) and there was also an association with development of metastases follow-up (logistic regression p=0.024). Using multivariate analysis (Cox regression) co-expression of nuclear beta-catenin and c-Myc turned out to be the strongest marker of impaired prognosis (p=0.001, HR 5.26, 95% CI 1.93-14.36). Expression of non-membranous gamma-catenin, cyclin D1 and c-Myc alone failed to have independent prognostic significance in our study.     

Malignant phyllodes tumour with a noninvasive ductal carcinoma component

 A malignant phyllodes tumour with a noninvasive ductal carcinoma component is reported. The patient was an 80-year-old Japanese woman with a breast tumour detected by routine physical examination. A simple mastectomy was performed. The excised tumour was 10.5×9.4×5.4 cm in size and bulged into the skin with ulceration. The macroscopic appearance was that of a phyllodes tumour. Histologically the tumour consisted mainly of stromal components with a leaf-like structure lined by atypical ductal epithelium. The epithelial component showed gradual evolution to intraductal papillary carcinoma in a few areas. The stromal component was composed mainly of fibrosarcoma with areas of osteosarcoma and rhabdomyosarcoma. Neither stromal invasion of intraductal carcinoma nor transition between the stromal and epithelial elements was seen. Three months after the operation, death occurred, with multiple pulmonary and subcutaneous metastases. This case probably represents malignant change in both the stromal and the epithelial components of a phyllodes tumour. Since the two elements were independent, the possibility that a phyllodes tumour may be one of the origins of true carcinosarcoma is raised. Received: 11 March 1997 / Accepted: 5 May 1997     

p53 expression in phyllodes tumours is associated with histological features of malignancy but does not predict outcome

AIMS: To study p53 protein expression in phyllodes tumours of the breast, with particular attention to its prevalence and to its relationship with histological features and clinical outcome. METHODS AND RESULTS: Stromal and epithelial p53 immunohistochemical expression was studied in 57 phyllodes tumours (27 benign, 17 borderline, 13 malignant) using an avidin-biotin peroxidase method. High levels of expression (> 30% of stromal nuclei) were found in eight phyllodes tumours (14%). p53 expression was associated with tumour grade (P = 0.001), prominent stromal overgrowth (P = 0.0003), prominent stromal nuclear pleomorphism (P = 0.006), high stromal mitotic count (P = 0.05), and an infiltrative tumour margin (P = 0. 05). Six patients were lost to follow-up after surgery. Mean follow-up time of the remaining 51 patients was 7.3 years (median 4. 3, range 0.5-25) or until death. Sixteen patients (31%) experienced tumour recurrence. Recurrence was more likely if there was an infiltrative tumour margin (P = 0.006) or prominent stromal overgrowth (P = 0.04) but not p53 expression (P = 0.55). A minority of recurrences expressed p53 more extensively than their primary counterparts. There were five tumour-related deaths (10% of patients). Death was associated with high grade (P = 0.0002), prominent stromal overgrowth (P = 0.0001), an infiltrative margin (P = 0.0002), prominent nuclear pleomorphism (P = 0.005), a high mitotic count (P = 0.01) and tumour size (P = 0.03). Again, p53 expression was not associated with tumour-related survival (P = 0. 13). CONCLUSIONS: p53 abnormalities occur in a minority of borderline and malignant phyllodes tumours. p53 expression is associated with known negative prognostic factors, but does not appear to be a useful determinant of tumour recurrence or long-term survival.     

Wnt pathway-related gene expression during malignant progression in ulcerative colitis

Long-standing ulcerative colitis (UC) has been associated with a high risk of developing colonic adenocarcinoma. Importantly, both low- and high-grade dysplasia are strongly related to the presence or development of malignancy. The canonical Wnt/beta-catenin signaling pathway is of crucial importance in cancer development and progression, but its role in UC-related carcinogenesis remains to be determined. We evaluated the immunolabeling patterns of beta-catenin, as well as the products of Wnt-associated cancer genes E-cadherin, cyclin D1 and c-myc, along the dysplasia-carcinoma pathway in UC. For this purpose, immunohistochemistry (IHC) was performed on 18 adenocarcinomas and 17 dysplasias, derived from 21 patients. We found that intracellular beta-catenin accumulation, the hallmark of Wnt signaling activation, is observed in dysplasia, together with enhanced labeling of nuclear protein cyclin D1 and reduction of membranous labeling of E-cadherin. c-myc displayed moderate immunolabeling in the (pre)malignant lesions. Thus, the Wnt pathway is activated in early stages of malignant progression in UC. Furthermore, upregulation of the oncogene cyclin D1 and downregulation of tumor suppressor E-cadherin also occurs in the (pre)neoplastic state. This may contribute to the high potential for malignant degeneration of dysplasia in UC-related colitis.     

Wnt/β-catenin信号通路在实验性大鼠肝癌形成过程中的作用

目的:探讨Wnt/β-catenin信号转导通路与肿瘤发生的关系,为肝癌的防治提供新的思路。方法:选取Wnt/β-catenin信号转导通路中上下游关键因子wnt1、β-catenin、APC、cyclinD1以及c-myc等,应用RT-PCR法观察它们在正常肝脏,不典型增生肝脏和肝癌组织中的转录水平。用免疫组化染色法研究β-catenin、APC和cyclinD1等3个因子有无表达。结果:在正常肝脏中,用RT-PCR未检测到wnt1、cyc-linD1以及c-myc的mRNA转录,免疫组化染色也只观察到β-catenin在细胞膜处有比较弱的表达。诱癌14周后,在发生不典型增生的肝组织中,检测到β-catenin、APC和cyclinD13个基因的转录。通过免疫组化染色也观察到β-catenin蛋白在胞质中的积累,APC和cyclinD1在部分细胞中出现表达。诱癌16周后,在肝癌组织中,除wnt1mRNA外,其他几个因子的mRNA都有转录,免疫组化也印证了上述各个发生转录的因子在蛋白水平都有不同程度的表达。结论:Wnt/β-catenin信号转导通路在大鼠的肝癌形成过程中被激活,其可能是实验性肝癌发生的原因之一。     

CD10, actin, and vimentin expression in breast phyllodes tumors correlates with tumor grades of the WHO grading system

The discrimination of borderline from malignant primary breast phyllodes (PT) tumor is still unclear. We studied 22 PT cases to investigate the immunohistochemical expression (staining of stromal CD10, SMA [smooth muscle actin], and vimentin) as well as the features of focal glandular atypia to determine whether these correlated with the histopathologic grading system. In our results, the stromal staining of CD10 was positive in 4 of 6 malignant and 2 of 5 borderline PT cases, but negative in all benign PT cases. Stromal actin and intraglandular vimentin-expressive tumor cells were found in 5 of 6 malignant PT cases but not in borderline and benign PT cases. There is a significant difference in the panel of stromal CD10, actin, and vimentin expression between borderline and malignant PT (p<0.05). Besides, the progression of malignant potential breast phyllodes tumor may cause glandular epithelium atypia with loss of polarity.     

Mammary fibroadenoma and some phyllodes tumour stroma are composed of CD34+ fibroblasts and factor XIIIa+ dendrophages

Fibroadenomas and mammary phyllodes tumour arise by proliferation of mammary stroma and epithelial elements. However, it is the stromal element that determines the biology of these biphasic tumours. Normal mammary stroma, like most collagenous connective tissue, contains resident populations of CD34+ dendritic interstitial cells and scattered factor XIIIa+ collagen-associated dendrophages. Actin + myofibroblasts are usually absent from mammary stroma in non-disease states. To determine whether CD34+ and factor XIIIa+ cells proliferate in fibroadenomas and phyllodes tumours, and to study myofibroblastic differentiation in these lesions, we examined 19 fibroadenomas in 14 patients along with five low grade and two high grade phyllodes tumours. We employed antibodies against the human progenitor cell antigen CD34, coagulation factor XIIIa and HHF-35 actin. In three fibroadenomas and two phyllodes tumours, we used Ki-67 antigen to study cell proliferation and oestrogen and progesterone receptors to study possible hormonal influence on stromal cells. In all fibroadenomas, CD34 strongly stained interlobular, pericanalicular and intracanalicular fibroblasts with collagenous and/or myxoid features. Four low grade phyllodes tumours also had CD34+ fibroblasts as did one high grade tumour. Actin reactivity varied and was most pronounced in six fibroadenomas resembling the so-called cellular variant, while seven regular fibroadenomas had no actin + stromal cells and six had only focal and weak actin + stromal cells. Factor XIIIa+ cells were prominently admixed in the stroma of all tumours studied comprising from 5% to 20% in fibroadenomas and, focally, up to 50% in phyllodes tumours. Oestrogen and progesterone receptors were expressed only in glandular elements. Ki-67 index in stromal cells was 1% to 3% in fibroadenoma, 10% to 20% in low grade, and 20% to 40% in high grade phyllodes tumour. We conclude that fibroadenomas and some phyllodes tumours are composed of CD34+ fibroblasts that show varying myxoid, collagenous or myofibroblastic differentiation. The fibroblasts are accompanied by a subset of dendritic histiocytes that express factor XIIIa. Fibroadenoma variants show prominent collagenous actin + myofibroblastic differentiation of CD34+ stromal cells, sometimes with a gradient of CD34 down-regulation. Fine-needle or limited stereotactic core biopsy of these biphasic tumours, if they yield only stromal cells, must be distinguished from other CD34+ stromal tumours. Increased factor XIIIa+ dendrophage populations were seen in phyllodes tumours, especially in two high grade tumours that had malignant fibrous histiocytoma-like features, suggesting clonal evolution toward the fibrohistiocytic final pathway. Further study of CD34 and factor XIIIa + mammary stromal cells in larger numbers of phyllodes tumours might ascertain whether increasing factor XIIIa reactivity correlates with differentiation and increased tumour aggressiveness.     

Significance of aberrant (cytoplasmic/nuclear) expression of beta-catenin in pancreatoblastoma

This study concerns the significance of aberrant (nuclear/cytoplasmic) expression of beta-catenin in pancreatoblastoma (PBL). On immunohistochemistry, all seven PBLs examined showed nuclear/cytoplasmic expression of beta-catenin, predominantly in the squamoid corpuscles (SCs). In areas with acinar/ductular differentiation, few tumour cells displayed nuclear/cytoplasmic expression of beta-catenin and more than half of the tumour cells showed membranous expression. Two out of five (40%) tumours examined showed missense mutations in codons 33 and 37 of exon 3 of the beta-catenin gene. No mutation of the adenomatous polyposis coli (APC) gene was detected in two of the remaining three tumours. Amplifiable DNA for APC analysis was not obtained from the one other tumour. Immunoreactivity for cyclin D1, one of the nuclear targets of beta-catenin, was found predominantly in the SCs of the seven tumours. In contrast, the Ki-67 labelling index was 2-4% (median 3%) in the SCs and 8-18% (median 12%) in the other areas, indicating a negative correlation with nuclear cyclin D1 reactivity. These results imply that in PBLs, nuclear/cytoplasmic accumulation of beta-catenin and overexpression of its target gene cyclin D1 are not associated with the induction of tumour cell proliferation. Nuclear/cytoplasmic accumulation of beta-catenin may be related to the morphogenesis of the SCs that are considered most characteristic for PBL.